Considerable evidence correlates high levels of polyamines with vigorous macromolecular synthesis and mitotic activity, and lower levels of polyamines with diminished macromolecular synthesis and aging. Is there a causal connection? We propose to investigate this question in systems wherein senescense and aging can be induced and prevented at will, and in which polyamines prevent aging. Excised leaves start to senesce immediately; this can be prevented by the appliation of cytokinin hormones (isopetenyladenine) or, as we have found, 1-10 mM polyamine. Evidence indicates that cytokinins act through elevated polyamine content. The aging syndrome involves a rapid rise in RNase activity (within 1 hr) and acid protease activity (6-8 hrs), diminished levels of protein and RNA, and ultimately (24-48 hrs), loss of pigmentation. All these events can be slowed or arrested by minute quantities of exogenous polyamines. These processes are best studied in isolated protoplasts. Here, exogenous polyamines stabilize the fragile plasma membrane, increase incorporation of precursors into DNA, RNA and protein, and stimulate mitosis, leading to numerous binucleate protoplasts. These cells contain ornithine decarboxylase (ODC), arginine decarboxylase (ADC), S-adenosylmethionine decarboxylase (SAMDC), lysine decarboxylase (LDC), and polyamine oxidase (PAO) activity. We have shown that the activity of the biosynthetic enzymes is inversely related to tissue age, and that ODC and ADC respond in expected ways to environmental and hormonal triggers that affect aging. We have also applied metabolite antagonists effective against ODC, ADC, SAMDC and PAO, and have noted changes in aging patterns and in polyamine content as determined by TLC of dansylated polyamines on silica gel and HPLC of benzoylated polyamines. We plan to compare protoplasts competent to divide (tobacco) and senescing, non-dividing protoplasts (oat). In each we will determine the quantity of polyamines and activity of relevant enzymes during natural and induced aging; control will be exerted by excision, hormones, inhibitors and polyamines themselves. Molecular mechanisms controlling rapid change in ODC and ADC activity will also be examined.